This application relates to an improved method for sequencing of nucleic acids using multiple fluorescent labels, and to apparatus and kits adapted for use with the method.
Sequencing of nucleic acids using the chain termination method involves the general steps of combining the target nucleic acid polymer to be sequenced with a sequencing primer which hybridizes with the target nucleic acid polymer; extending the sequencing primer in the presence of normal nucleotide (A, C, G, and T) and a chain-terminating nucleotide, such as a dideoxynucleotide, which prevents further extension of the primer once incorporated; and analyzing the product for the length of the extended fragments obtained. Analysis of fragments may be done by electrophoresis, for example on a polyacrylamide gel.
Although this type of analysis was originally performed using radiolabeled fragments which were detected by autoradiography after separation, modern automated DNA sequencers generally are designed for use with sequencing fragments having a fluorescent label. The fluorescently labeled fragments are detected in real time as they migrate past a detector.
U.S. Pat. No. 5,171,534 which is incorporated herein by reference describes a variation of this basic sequencing procedure in which four different fluorescent labels are employed, one for each sequencing reaction. The fragments developed in the A, G, C and T sequencing reactions are then recombined and introduced together onto a separation matrix. A system of optical filters is used to individually detect the fluorophores as they pass the detector. This allows the throughput of a sequencing apparatus to be increased by a factor of four, since the four sequencing reaction which were previously run in four separate lanes or capillaries can now be run in one.
It is an object of the present invention to provide a further improvement for use with chain termination sequencing reactions which can further increase the throughput of an instrument.
In order to use nucleic acid sequencing as a diagnostic tool, it will be necessary to determine the sequence of the same DNA region from many samples. The present invention makes it possible to increase the throughput of an instrument being used for this purpose. Thus, a first aspect of the invention provides a method for evaluating the sequence of a target nucleic acid polymer in a plurality of samples. In this method, each sample is first divided into four aliquots which are combined with four sequencing reaction mixtures. Each sequencing reaction mixture contains a polymerase enzyme, a primer for hybridizing with the target nucleic acid, nucleotide triphoshate feedstocks and a different dideoxynucleotide triphosphate. This results in the formation of an A-mixture, a G-mixture, a T-mixture and a C-mixture for each sample containing product oligonucleotide fragments of varying lengths. The product oligonucleotide fragments are labeled with fluorescent tags, and these tags will generally be the same for all four sequencing reactions for a sample. However, the fluorescent tags used for each sample are distinguishable one from the other on the basis of their excitation or emission spectra.
Next, the A-mixtures for each sample are combined to form a combined A mixture, the G-mixtures are combined to form a combined G-mixture and so on for all four mixtures. The combined mixtures are loaded onto a separation matrix at separate loading sites and an electric field is applied to cause the product oligonucleotide fragments to migrate within the separation matrix. The separated product oligonucleotide fragments having the different fluorescent tags are detected as they migrate within the separation matrix.
The method of the invention can be used as described above to determine the position of every base in the sequence, or it can be used to determine the position of less than all four bases. For example, the method can be used to determine the position of only the A bases within a sequence for some diagnostic applications.
A further aspect of the present invention is a kit useful for diagnostic sequencing of a selected portion of a gene. One embodiment of such a kit contains a plurality of sequencing primers for the selected portion of the gene, each sequencing primer being identical in its DNA sequence but being labeled with a different fluorescent tag.
A further aspect of the invention is an apparatus for performing the method of the invention. Such an apparatus comprises
(a) means for providing excitation energy to a detection site within a separation matrix disposed within the apparatus;
(b) means for detecting light emitted from fluorescently-labeled oligonucleotide fragments located within the detection site;
(c) configuration control means, operatively connected to the means for providing excitation energy and the means for detecting to provide combinations of excitation wavelength and detection wavelength specific for a plurality of different fluorescently-labeled oligonucleotide fragments; and
(d) data processing means, operatively connected to the configuration control means and the means for detecting for receiving a signal from the means for detecting and assigning that signal to a data stream based upon the combination of excitation wavelength and detection wavelength set by the configuration control means.